Particulate matter (PM) air pollution is a global environmental health problem that causes 800K premature deaths per year worldwide largely due to increased acute thrombotic cardiovascular events. This team has discovered that PM induces a prothrombotic state and accelerates vascular thrombosis via mitochondrial ROS-dependent release of IL-6 from lung macrophages. While using a standard reductionist approach data was generated to show that mitochondrial ROS-induced release of IL-6 plays a key role in PM-induced toxicity. This data and that from other labs suggest that PM alters gene transcription through complex pathways, including epigenetic modifications. However, it is not known whether PM induces epigenetic changes via mitochondrial ROS. Dynamic chemical modifications of DNA by cytosine methylation (5mC) and hydroxymethylation (5hmC) represent a fundamental mechanism of biological regulation. In preliminary studies in alveolar macrophages, it was observed that PM causes loss of DNA methylation characterized by a reduction in 5mC and an increase in 5hmC, which could be reversed by an antioxidant. It was also found that PM increased Tet1 but decreased DNMT1 expression in alveolar macrophages. To specifically examine the links between mitochondrial ROS and DNA and RNA methylation in alveolar macrophages, a transgenic mouse was engineered that expresses a mitochondrial-targeted redox sensitive GFP (mito-roGFP), which allows one to isolate macrophages in which exposure to physiologically relevant concentrations of inhaled PM has increased the generation of mitochondrial ROS and compare them with unaffected macrophages. In the R21 phase, this tool will be exploited to test the hypothesis that PM-induced mitochondrial ROS alter IL-6 DNA and RNA methylation to regulate expression and determine whether PM-induced mitochondrial ROS regulate DNA/RNA methylation of the il-6 gene and its transcriptional regulators in alveolar macrophages (R21 Aim 1) in vitro and (R21 Aim 2) in vivo. The increased resources in the R33 phase will allow the analysis to be expanded to examine genome- and transcriptome-wide changes in DNA and RNA methylation patterns. To demonstrate causality, in the R33 phase, these changes will be measured in animals treated with mitochondrial-targeted antioxidants. To demonstrate the biologic importance of these findings, this approach will be used to determine whether the stochastic replacement of tissue-resident macrophages with monocyte-derived macrophages over the lifespan might explain why some individuals are more susceptible to PM-induced toxicity. Using a unique toolset to genetically label these different alveolar macrophage populations, the hypothesis that these distinct macrophage populations respond differentially to PM in their mito- ROS production, and DNA/RNA methylation profiles will be interrogated. In the R33 Aim 1, a determination of whether mitochondrial ROS are required for PM-induced changes in genome-wide DNA and transcriptome-wide RNA methylation in macrophages in vivo will be performed. In Aim 2, an examination as to how PM-induced mitochondrial ROS and changes in DNA/RNA methylation profiles differ in tissue-resident and monocyte-derived alveolar macrophages will be addressed.